Vasylyna-Marta Tseduliak (Lviv / UA), Oksana Koshla (Lviv / UA), Bohdan Ostash (Lviv / UA), Andriy Luzhetskyy (Saarbrücken / DE), Satoshi Matsumoto (Sendai / JP), Yoshiyuki Ohtsubo (Sendai / JP), Yuji Nagata (Sendai / JP)
Introduction
Well-known antibiotic-producing genus Streptomyces remains a source of hidden chemical diversity. The introduction of point mutations in the rpsL gene for r-protein
S12 is one of the tools that has shown great potential for manipulating the metabolism of these bacteria. One of the currently most popular heterologous
expression hosts, Streptomyces albidoflavus J1074, was chosen to study the effect
of the engineered rpsL mutations (1).
Goals
This work aims to study the properties of rpsL mutants K88E, K88R, and R94G.
Methods
Scanning electron microscopy; CFU, dry biomass, protein content measurement in TSB medium; fermentation in R5 and SDB media and subsequent antibiotic
extraction; c-di-GMP extraction from TSB; HPLC-MS; bioactivity assays; qPCR; RNA-seq.
Results
Strains S. albidoflavus K88E and K88R exhibited altered morphology and a reduced number of sporulating hyphae compared to SAM2 and R94G. Similar levels of total protein accumulation and CFU counts were observed in all strains. However, the dry biomass weight of the original strain SAM2 was significantly increased. The c-di-GMP level, a key regulator of specialized metabolite production, was notably reduced in the K88R mutant, and all strains showed a decrease in the expression of genes for c-di-GMP synthesis. K88E and R94G produced, on average, more specialized metabolites than SAM2 and K88R, as confirmed by bioassays and HPLC-MS analysis of extracts.
Summary
The rpsL mutants exhibit distinct morphological and growth differences, highlighting the pleiotropic effects of missense mutations within genes for ribosomal proteins. To the best of our knowledge, our work for the first time links rpsL mutations to c-di-GMP metabolism in streptomycetes. This offers fresh insight into the mechanisms of pleiotropicity of rpsL mutations.
References
1. Lopatniuk M, Myronovskyi M, Nottebrock A, Busche T, Kalinowski J, Ostash B, et al.
Effect of "ribosome engineering" on the transcription level and production of S. albus
indigenous secondary metabolites. Applied Microbiology and Biotechnology. 2019 Sep
19;103(17):7097–110.